Use of quillaja saponaria extracts for the prevention and control of viral infections in fish

ABSTRACT

The present invention provides a new use of Quillaja extracts for the manufacture of a medicinal composition for the prevention and control of viral diseases in fish, which is preferably administered orally in combination with food. Other objects of invention relate to a medicinal composition and a food composition for the prevention and control of viral diseases in fish comprising the described Quillaja extracts.

TECHNICAL FIELD

The present invention relates to the aquaculture industry, and particularly provides a new use of extracts of the plant Quillaja saponaria for the prevention and control of viral diseases in fish, as well as compositions comprising said extracts.

BACKGROUND OF THE INVENTION

Salmon farming industry has grown significantly worldwide in the last two decades; particularly in Chile this economic sector has become one of the most important for the country. However, different viral diseases infect fish and have a negative effect on production (Tobar, I. et al. (2015). Frontiers in Immunology, (6) 244).

Among the viral diseases affecting fish, infectious pancreatic necrosis (IPN) is a highly contagious disease affecting farmed salmonids which causes high mortality rates in fry during their first feeding stage and in juveniles (smolts) after transfer to seawater. This disease is one of the most important diseases in Chile and is considered endemic in the country, causing great economic losses in the salmon industry (Tapia, losses D. et al. (2015). Diseases of Aquatic Organisms, 116 (3), 173-184). Its etiologic agent is the IPN virus (IPNv). Currently, the IPNv is one of the pathogens most frequently detected by diagnostic laboratories in marine and freshwater farms, and is the second leading cause of mortality in adult Atlantic salmon in fish farms in Chile (Sernapesca (2013). Informe sanitario de salmonicultura en centros marinos 2012, Servicio Nacional de Pesca y Acuicultura. Valparaiso). However, the geographical distribution of this pathogen is not limited to Chile and it has been historically found in Norway (Diseases of Aquatic Organisms, 114 (3), 177-187 Jensen, B. et al. (2015).) and more recently in steelhead trout in South Africa (doctoral Dissertation Bragg, R R (2015). Isolation and Identification of Infectious Pancreatic Necrosis Virus from Rainbow trout (Salmo gairdneri Richardson) in South Africa), reaching a worldwide distribution. Given the negative impact on the health of fish caused by this virus, there is a constant need to seek alternatives for its control.

The virus that causes infectious pancreatic necrosis belongs to the Birnaviridae family and Aquabirnavirus genus, and it is characterized by having a non-enveloped capsid with a genome consisting of double stranded RNA. Clinical symptoms of infection include swelling of the abdomen and eyes, skin darkening, necrosis of the pancreatic tissue and spiral swim; which can cause death of fish. In the juvenile stage of freshwater, an outbreak of IPNv can cause 100% mortality. To prevent this viral agent various vaccination strategies have been provided, however, IPNv outbreak control depends on the biosafety of the farms and the level of resistance of fish (Robledo, D. et al. (2016). BMC Genomics, 17 (1), 1).

Another viral agent in the aquaculture industry is the infectious salmon anemia virus (ISA) which attacks mainly salmonids and can lead to high mortality rates in the population of affected fish, threatening the main areas of fish farming in the North Atlantic Ocean and in Chile. The ISA virus (ISAv) belongs to the Orthomyxoviridae family, Isavirus genus and has a single-stranded RNA genome with envelope (Vike, S. et al. (2014). Aquaculture, 420, 119-125). The virus inside the fish infects all organs but preferably the endothelium, causing bleeding, lethargy, abdominal distension and severe anemia in the affected fish. The mortality caused by this disease is high and few fish remain alive as carriers. In Chile, the first outbreaks occurred in mid-2007 and the solution was the elimination of all salmon infected.

Currently, due to the nature of the virus, control through the use of medicinal compounds is not effective or economically viable and prevention measures consist of maintaining a strong biosafety (imported egg control), use of vaccines and total elimination of sea-cages infected.

Multiple strategies to increase the resistance of fish to pathogens have been used in parallel with the development of vaccines. As an alternative, the administration of food for fish that improves health has been used. In this regard, diets rich with microalgae containing polyunsaturated fatty acids, glycans, carotenoids, among others have been used. These ingredients can promote fish welfare, improve intestinal health and increase resistance to disease (Kousoulaki, K. et al. (2015). Journal of Nutritional Science, 4, e24).

Quillaja saponaria Molina (common name Quillay) is a native tree of Chile primarily used as a soap substitute due to the presence of saponins (San Martin, R. (1999). Economic Botany, 53 (3), 302-311). Saponins can be obtained industrially as powder or liquid extracts, and may be in a purified state, partially purified or unpurified. These extracts are marketed by several companies, being one of the most important Natural Response and Desert King (San Martin, R. and Briones, R. (2000). Journal of the Science of Food and Agriculture, 80 (14), 2063-2068).

To date, the extracts rich in saponins are used as natural emulsifiers in cosmetics, food and beverages. Additionally, these are used as adjuvants for vaccine production and pharmaceutical formulations (Maier, C. et al. (2015). Journal of Agricultural and Food Chemistry, 63 (6), 1756-1762). Other uses have also been as a biocide to eliminate nematodes (US 20050074508), mollusks (US 20070196517) and fungi (Elizondo, E. A. M. et al. (2010). Agro south, 38 (2), 87-96).

In the review of Wang, Y. et al. (2016), International Journal of Molecular Sciences, 17 (3), 325, it is described that saponins can modulate the immune system of shrimp and fish, and promote the growth of the latter. However, most saponins are unstable in aqueous conditions and are very toxic to fish at high concentrations.

Prior art analysis regarding the application of Quillaja extracts in fish, showed patent application PCT WO2015155293 that discloses an oral food additive for use in the prevention and/or treatment of infections and particularly describes a composition comprising Quillaja saponaria saponins for prophylactic treatment of the ectoparasite copepod Caligus in fish. The experimental evidence provided by this document does not sustain the beneficial effect against other pathogens.

Patent application WO2015179840 describes combinations or compositions comprising Yucca schidigera and Quillaja saponaria, and further include antimicrobials, antibiotics and anticoccidial agents, for administration to animals to prevent diseases. As a general disclosure, it describes that can be applied to fish orally.

Patent WO0151083 application discloses the adjuvant composition comprising a saponin and an oligonucleotide comprising at least one CpG unmethylated dinucleotide. Preferably, the composition includes saponins derived from Quillaja saponaria, and most preferably, the saponin is chemically modified or includes a substantially pure saponin (QS-7, QS-17, QS-18 or QS-21). No description is done for the use in fishes.

Chilean patent application CL200402942 discloses a food additive fish formulated with purified extract of Quillaja saponaria Molina comprising 15-25% w/w of triterpene saponins obtained from the extract and 75-85% w/w of potato maltodextrin. This document does not mention that this food promotes the health of the fish.

The PhD thesis of Fernandes, R. N, (2014) Using Quillaia saponin (Quillaja saponaria Molina) em juvenis of pacu, Universidade Estadual Paulista, Faculty of Agricultural Sciences and Veterinary Center Aqüicultura, Brazil, describes a study wherein the effect of administering Quillaja saponins in doses from 100 to 400 mg/Kg in pacu fish (Piaractus mesopotamicus). Following 15 days of feeding fish with Quillaja saponins, 325 fish were inoculated with Aeromonas hydrophila and clinical signs were observed. After seven days, the survival of pacu fish against experimental infection was higher in fish fed with Quillaja saponins in a dose of 200 mg/Kg.

On the other hand, Vinay et al. (2014), Veterinary immunology and immunopathology, 158 (1), 73-85, describes an evaluation of the systemic effect of Quillaja saponins administered intraperitoneally as vaccine adjuvant in Paralichthys olivaceus. This study showed that saponins are good inducers of inflammation, but are also toxic to the fish. Saponins concentrations of 500, 160, 50, 16 and 5 μg/fish produced 95%, 65%, 20% and 5% mortality rates, respectively, with a lethal dose (LD₅₀) of 22.4 mg/Kg. The results determined that the toxic effect of saponins depended on the level of purification and the source of the product. Finally, the authors found that a concentration of 3.4 mg/Kg of fish is toxic when is administered intraperitoneally, and it is recommended to use a lower concentration in Paralichthys olivaceus.

Regarding disclosures of saponins from sources other than Quillaja saponaria, Krogdahl et al. (2015), Journal of Agricultural and Food Chemistry, 63 (15), 3887-3902 discloses that soybean saponins administered orally as feed additive in doses of 2-10 g/Kg produced intestinal inflammation in Atlantic salmon (Salmo salar), and the severity is dose-dependent.

This analysis of prior art shows that, although Quillaja saponaria saponins have been described for various uses and applications, they are not associated with pathogens of interest in commercial fish.

SUMMARY OF THE INVENTION

An object of the present invention relates to the use of a Quillaja saponaria extract for manufacturing a medicinal composition for the prevention and control of viral disease in fish. The medicinal preparation for fish is administered orally in a dose range of 0.9 to 12 mg/Kg of saponins per live weight. In a preferred embodiment, administration to fish is done orally in combination with food. Viral diseases are caused by a virus belonging to the families of the group comprising Birnaviridae, and Orthomyxoviridae, genus Aquabirnaviridae and Isavirus, respectively; and in particular for the viruses of the pancreatic necrosis and infectious salmon anemia, respectively.

The group of fish intended for the present invention are preferably salmonids, where these are selected from the group consisting of Salmo salar, Salmo trutta, Salmo gairdnerii, Oncorhynchus mykiss or Coho salmon species.

Another object of the present invention is a medicinal composition for the prevention and control of viral diseases in fish, comprising a Quillaja saponaria extract and an appropriate excipient. Quillaja saponaria extract is in a ratio of 2 to 90% w/w of the total weight of the composition.

The present invention also provides a food composition for the prevention and control of viral diseases in fish, comprising a Quillaja saponaria extract and an appropriate additive. Quillaja saponaria extract is in a proportion of less than 6% w/w of the total weight of the composition.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is the chromatographic profile of a complete Quillaja saponaria Molina extract measured by HPLC, where the main saponins QS7, QS17, QS18 and QS21 are indicated.

FIG. 2 is an image obtained by optical microscopy of ASK salmonid cell line in the presence of different concentrations of Quillaja extracts UD100-Q (Ultra Dry® 100-Q) and QD100 (Quillaja Dry® 100).

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a novel use of plant extracts of Quillaja (Quillaja saponaria) for the prevention and control of viral infections that affect fish. The inventors have found that different extracts of Quillaja saponaria Molina saponins, a Chilean endemic tree, have a protective effect against viral infections affecting salmonid farming. The inventors have tested and acquired appropriate dose concentrations of Quillaja extracts to be used safely in salmon, without altering pathophysiological treated individuals.

The present invention relates to the use of Quillaja extracts, which have a specific profile of saponins. In all cases, the profiles of these extracts are saponins exclusive of Quillaja saponaria, either in purified extracts, partially purified or unpurified.

Total or unfractionated extracts of Quillaja saponaria Molina have a distinctive profile and own saponins containing over 100 types of chemically different saponins. The main saponins are QS7, QS17, QS18 and QS21, as seen in the chromatographic profile of FIG. 1. The relative concentrations of these saponins depend on the source of the raw material. Additionally, the partially purified extracts (from 2 to 90% w/w or w/v of saponin as powder or liquid) contain non-saponin compounds which mainly include a mixture of polyphenols and, in smaller amounts, other sugars.

Extracts of Quillaja saponins can be obtained industrially as powder or liquid extracts, with varying degrees of purification. For example, for the purposes of the present invention, various commercial extracts of Quillaja saponaria may be used, such as those shown in Table 1 below.

TABLE 1 Commercial extracts (Desert King Chile) of Quillaja saponaria useful for the present invention. Product name Description Ultra Dry ® Quillaja saponaria Molina powder extract, mainly containing 100-Q triterpene saponins up to 65% w/w. Quillaja Dry ® Quillaja saponaria Molina powder extract, mainly containing 100 triterpene saponins up to 25% w/w. Vax Sap  ® Highly purified Quillaja saponaria Molina powder, mainly containing triterpene saponins >90% w/w. QL 1000  ® Liquid extract mainly of Quillaja saponaria Molina at a concentration of 8% w/v of saponins. QL Perm  ® Liquid extract mainly of Quillaja saponaria Molina at a concentration of 2% w/v of saponins.

The present invention discloses the use of Quillaja saponaria extract to develop a medicinal composition and a food composition for the prevention and/or control for undesired medical conditions related to viral diseases in fish. The new use of Quillaja saponaria extracts described in the present invention can serve to obtain drugs intended to prevent, control, cure, treat or alleviate viral diseases in fish; and also can be obtained for medicinal use in food products that have the same therapeutic purposes.

The present invention encompasses the use of Quillaja saponaria extracts for the prevention of viral infections caused by non-enveloped virus, such as IPNv or others affecting salmonid fish. In particular, infectious pancreatic necrosis (IPN) is a highly contagious viral disease affecting fish of all species of salmon. The most susceptible species are rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), brown trout (Salmo trutta), Atlantic salmon (Salmo salar), and Pacific salmon (Oncorhynchus spp.). The stages most affected by this viral disease are fingerlings, but also the disease occurs in young or Atlantic salmon smolts within the first weeks after transfer from fresh water to seawater. The classic signs of outbreaks of IPN are a sudden increase, and usually progressive, daily mortality, accompanied by clinical signs such as increased dark pigmentation, distended abdomen and corkscrew swimming motion. IPNv also causes disease in other species of food fish such as yellowtail (Seriola quinqueradiata), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus) and Atlantic cod (Gadus morhua), which also can be treated with Quillaja saponaria extracts for medicinal purposes, and in general on any fish species susceptible to infection of IPNv.

The present invention also encompasses the use against enveloped viruses, being the infectious salmon anemia virus the most important (ISAv). Fishes infected with this virus show clinical signs that may include lethargy, anemia, leucopenia, ascites, exophthalmos, skin darkening and high mortality. Severe anemia is usually associated with very pale gills. ISAv outbreaks occur primarily in Atlantic salmon (Salmo salar) but infections have been reported in other species such as Coho salmon (Oncorhynchus kisutch) and experimentally in rainbow trout (O. mykiss). Natural reservoirs of the virus are river trout (Salmo trutta), chum salmon (O. keta), chinook salmon (O. tshawytscha) and Arctic char (Salvelinus alpinus). Also the virus affects other non-salmonid species, such as herring (Clupea harengus), Atlantic cod (Gadus morhua) and pollack (Pollachius virens), where Quillaja saponaria extracts can also be used for medicinal purposes and generally any kind of susceptible fish to infections by the virus.

Quillaja saponaria extracts in the present invention are used to elaborate a medicinal product or a food product for the prevention or post-infection control of virus in fish. Such products are orally administered in solid or liquid form.

For the use of Quillaja saponaria extracts in the preparation of a composition or medicinal preparation, medicine or veterinary composition, different formulations containing such extracts as active ingredient can be used. Quillaja saponaria extracts are in a proportion between 2% and 90% w/w of the total weight of the composition. The pharmaceutical form can be powder, oil suspension or as part of a food pellet. In a preferred embodiment, the medicinal composition comprises veterinary suitable excipients for oral administration in salmonids, such as lactose, corn starch, silicon dioxide, among others well known by experts of this area.

For the use of Quillaja saponaria extracts in the preparation of a product or food composition or dietary supplement, these extracts are mixed with fish food in a pellet form, which is mixed with an equivalent of 2% of oil/weight of the pellet to impregnate, to adhere the extract to the food. Examples of fish diets widely known in the industry are produced by Ewos, BioMar, Salmofood, among others. In a preferred embodiment, the food composition comprises food additives appropriate for oral administration in salmonids, such as fishmeal, fish and/or vegetable oil, vitamins, minerals, among others well known by experts of the area.

Examples of implementation of the invention have been included for the purpose of illustrating the invention, with the preferred embodiments and comparative examples, but in no case to be considered as a restriction to the scope of the patent application, which it is only delimited by the content of the claims appended hereto.

EXAMPLES Example 1: Evaluation of In Vitro and In Vivo Toxicity of Quillaja Extracts In Vitro Citotoxicity Assay in Salmon Cell Lines.

Assays with Quillaja extracts products were tested on cell monolayers derived from salmon in order to assess the citotoxicity. The cell lines used were SHK-1 and ASK. SHK-1 line, described as macrophage-like cells (Salmo salar; ECACC 97111106 Number, European Collection of Cell Culture, Salisbury, Wilts, SP4 0JG, UK) was cultured at 15° C. in Leibovitz 15 medium (L-15, Gibco, Invitrogen, Carlsbad, Calif., USA) supplemented with 10% v/v fetal bovine serum (Hyclone, Thermo Fisher Scientific, Logan, Utah, USA), 4 mM L-glutamine (Gibco), 1% v/v 2-mercaptoethanol (2-ME, Gibco) and 50 μg/mL gentamicin (US Biological, Swampscott, Mass., USA). The cell line ASK (Atlantic Salmon Kidney, ATCC® ™ CRL2747) was cultured at 16° C. in Leibovitz (L-15, Hyclone, Thermo Scientific), supplemented with gentamicin (50 μg/mL), L-glutamine (4 mM) (Gibco, Thermo Scientific), 2-mercaptoethanol 1% (v/v) (2-ME, Gibco) and 10% fetal bovine serum (v/v) (FBS, Hyclone).

All Quillaja extracts products were prepared in MEM or 15 Leibovitz medium at a concentration of 1 mg/mL, being dissolved at 37° C. for 3 hours with gentle stirring. All prepared solutions of these extracts were filtered through a 0.22 μm nitrocellulose membrane of to avoid contamination in cell cultures. The evaluated dilutions were prepared by serial dilutions from the standard solution.

To assess the cytotoxicity of Quillaja extracts in salmonid cell lines, 5×10⁵ cells/well were seeded in 6-well plates and incubated in 2 mL of culture medium as final volume for 72 hours at 15° C. After this time the culture medium was replaced with fresh medium and the confluency was verified. After 24 hours, cells were incubated with the different Quillaja extracts in 1 mL of culture medium. Cytotoxicity assessment was made after 24 hours incubation with Quillaja extracts. For this, the cells were washed twice with cold PBS and then disaggregated using a solution with 0.05% trypsin and 0.02% EDTA. Cells were analyzed by flow cytometry (FACS Canto II (Becton Dickinson) and incorporation of propidium iodide was determined as a marker for dead cells. Cells were incubated with a solution of ethanol as a positive control of cell death. As negative control, cells were incubated without Quillaja extracts, but were subjected to the same conditions. Additionally, cytotoxicity was assessed by visualizing cells by light microscopy.

Results indicated that the concentration that exhibited a 50% of cell death (CC₅₀) was between 3.5 and 83.4 μg/mL and the CC₉₀ varied between 4.7 and 92.6 μg/MI, depending on the product used as indicated in Table 2. FIG. 2 shows representative results with products QD 100 (Quillaja Dry® 100) and UD 100Q (Ultra Dry® 100-Q) by observing the cell monolayer through optical microscopy.

TABLE 2 Cell citotoxicity (CC₅₀) by flow citometry using propidium iodide. Product CC₅₀ in SHK-1 (μg/mL) CC₉₀ in SHK-1 (μg/mL) Vax Sap  ® 20.4 25.3 Ultra Dry ® 100-Q 22.1 29.2 Quillaja Dry ® 100 83.4 92.6 QL 1000  ® 3.7 6.5 QL Perm  ® 3.5 4.7

In Vivo Citotoxicity Assay in Fish.

To determine the short term oral toxicity (60 days), 550 Atlantic salmon (Salmo salar) clinically healthy fishes were taken, with an average weight of 9.5 g. Prior to the experiment the fish were acclimated for 8 weeks, during which 50 fish were randomly examined to check health condition through necropsy and microbiological tests to verify the absence of pathogens such as viruses, bacteria and parasites (Thoesen J. (1994) Suggested procedures for the detection and identification of finfish and shellfish Certain pathogens, 4th edn. Fish Health Section, American Fisheries Society, Bethesda, Md.; OIE (Office International des Epizooties) (2000) Diagnostic Manual for aquatic animal diseases, 3rd edn. OIE, Paris).

Fish were held in 1,000 L capacity fiberglass tanks each with independent water supply. The level of dissolved oxygen in the water was 10 mg/L. Water temperature, and oxygen levels of nitrogen compounds were controlled daily.

Extruded feed pellets (Micro 10, prepared by Ewos) were used to prepare five diets with 0, 100, 200, 300 and 600 ppm of saponins/Kg of food. These doses are equivalent to 0, 2, 4, 6 and 12 mg of saponins/Kg of live weight of fish, respectively. The fish were divided into 10 individual tanks with 50 fish each (5 groups with duplicate).

Fishes were hand fed twice a day with diets according to the expected live weight and growth rate of fish. To do this the weight of fish where obtained at 0, 30 and 60 post-start of the experiment.

Fish were observed at least three times daily during the study, recording any possible clinical signs and mortalities. The experiment lasted 60 days. Results during the course of the trial showed no mortalities or abnormalities attributable to the product administered at the different doses. Additionally, no macro or microscopic pathological alterations in the liver or intestine in any treated group, compared to the control group, were found. In conclusion, Quillaja extracts were safe for administration at tested doses.

Example 2: Use of Quillaja Extracts for the Prevention and/or Treatment of In Vitro Viral Infections in Fish Antiviral Activity of Quillaja Extracts Against IPNv.

The antiviral activity of the extracts was measured through infection assay in CHSE-214 cell monolayers derived from salmon (Oncorhynchus tshawytscha, ATCC Number CRL-1681, American Type Culture Collection). This cell line was grown at 16° C. in culture medium (MEM, Gibco) supplemented with 10% Fetal Bovine Serum (Hyclone), 2 mM L-glutamine (Gibco), 10 mM HEPES (Hyclone), 100 IU mL- 1/100 ug/mL-1 of gentamicin (Gibco). The CHSE-214 cells were incubated in culture plates with 24 well plates at a confluence of 90%. To determine the antiviral activity, the culture medium was removed and the monolayer was infected with a viral suspension of a Chilean IPNv isolated with an approximate 50 plaque forming units (PFU) and different Quillaja extracts. After 1 hour of viral adsorption at 15° C., the inoculum was removed and the cell monolayer was covered with agarose gel of low temperature of gelation at 0.5% in growth medium supplemented with different Quillaja extracts. It was incubated for 3 days at 15° C. and then the cells were fixed with 1 mL of 37% formaldehyde at room temperature for 1 hour. After removing the fixative and the agarose overlay, the cell monolayer was stained with 0.5% crystal violet solution for 1 hour. Finally, cells were washed with water and count of plaque forming units (PFU) was done. The efficiency of infection was quantified by the number of PFU obtained and compared with the percentage of the untreated control. Each condition was performed in triplicate.

Results showed that Quillaja extracts could effectively control the infection against the IPN virus, which is shown in Table 3. Of the extracts tested, VaxSap, UD100Q and QD100 were highly effective at doses of 1.09; 1.46 and 0.73 μg/mL, respectively. Furthermore, it was observed that with a low concentration of saponins, 40% of uninfected monolayer (QL Perm1 and Perm 2) was obtained.

TABLE 3 Efficacy (% uninfected cell layer) of Quillaja extracts concentrations according to products and saponin content in in vitro infections with IPNV. Product Saponin concentration concentration Non-infected Product (μg/mL) (μg/mL) monolayer (%) Positive 0 0 0 ^(c) Control VaxSap 1.22 1.09  75 +/− 3.9 ^(a) UD 100 Q 2.44 1.46  69.5 +/− 1.3 ^(ab) QD 100 2.44 0.73 59.1 +/− 1.2 ^(b) QL 1000 0.78 0.0078 0 ^(c) QL Perm 1 0.78 0.0078 43.9 +/− 2.2 ^(d) QL Perm 2 0.78 0.0078 45.8 +/− 1.4 ^(d) Note: ^(a), ^(b), ^(c) and ^(d) denote statistical differences at p < 0.05

Antiviral Activity of Quillaja Extracts Against ISAv.

Viral infection was carried out using approximately 1×10⁶ cells of ASK cell line [Atlantic Salmon were sown. Kidney] (ATCC® CRL2747 ™), which grew into a 6-well plate to a confluence of 80 to 90%. The cells were cultivated at 16° C. in Leibovitz medium (L-15, Hyclone, Thermo Scientific), supplemented with gentamicin (50 μg/mL), L-glutamine (4 mM) (Gibco, Thermo Scientific), 2-mercaptoethanol 1% (v/v) (2-ME, Gibco), fetal bovine serum 10% (v/v) (FBS, Hyclone). To determine the antiviral efficacy, cells were incubated for 4 hours with the viral inoculum (Chilean isolate a viral titer of 10⁶ copies of viral RNA). Subsequently, the culture medium was removed and fresh culture medium supplemented with antibiotics and different Quillaja extracts was added. Infectivity was quantified by qRT-PCR technique. This technique allowed the quantification of the number of copies of viral RNA obtained as a result of a viral infection. Results were expressed as a percentage comparison between the number of copies produced in the untreated condition versus treated Quillaja extracts, according to Table 4. Each condition was performed in triplicate. All Quillaja extracts were able to control the in vitro infection above 95%.

TABLE 4 In vitro efficacy (viral proliferation decrease) of different concentrations of Quillaja extracts according to products and saponin content, against ISAv. Product Saponin Viral concentration concentration copies/mL Viral growth Product (μg/mL) (μg/mL) (10⁴) inhibition (%) Positive 0 0 870,818 +/− 15,903   0 ^(a) control VaxSap 0.48 0.43 0.0005 +/− 0.0003 >99 ^(b) UD 100 Q 0.48 0.31 44,800 +/− 24,335  95 ^(b) QD 100 0.48 0.12 0.010 +/− 0.007 >99 ^(b) QL 1000 7.8 0.78 0.006 +/− 0.003 >99 ^(b) QL Perm 1 7.8 0.078 0.010 +/− 0.007 >99 ^(b) QL Perm 2 7.8 0.078 0.046 +/− 0.003 >99 ^(b) Note: ^(a), ^(b), ^(c) and ^(d) denote statistical differences at p < 0.05

Example 3: Preparation of a Fish Feed Impregnated with Quillaja saponaria Extracts

Quillaja extracts were mixed with fish oil to achieve the desired concentrations and then this mixture was impregnated at a rate of 4.9:1 (oil:dry pellet) in fish feed. This was done using an industrial mixer and incorporating oil in the food in movement for at least 5 minutes additional mixing for at least 10 minutes.

To obtain a food composition impregnated with Quillaja extracts at the effective dose, important parameters to be considerated are saponin doses/live weight of fish to be administered in the range from 0.9 to 12 mg of saponins per kilogram of live weight, fish weight and the amount of food they consume. The percentage of saponins in each particular Quillaja extract product should also be noted beforehand. Thus, any skilled person can obtain the ratio of saponins to be incorporated in the food.

As an example, to prepare a food composition considering the QL Perm® product, it must be used in a proportion of 6% w/w (60 g of product per kilogram of fish food).

Example 4: Use of Quillaja Extracts for the Prevention and/or Treatment of Viral Infections in In Vivo In Vivo Efficacy of Quillaja Products Against IPNv

Two hundred and forty Salmo salar fry with an average weight of 5 to 6 g were used. Selected fishes had no history of ISAv, which was checked by sampling and subsequent analysis of molecular diagnosis by RT-PCR in real time. Additionally, fishes were checked for bacterial and viral diseases. Before transferring the fishes to the experimental station, 60 fish were randomly sampled to determine their health condition, which considered necropsy, gill inspection, intestine and skin sampling, Gram staining in internal organs (spleen, kidney and brain), staining with acridine orange in gills, IFAT analysis for BKD and SRS, and RT-PCR for IPNv. The general clinical appearance of the fish was acceptable and was within normal parameters for the salmon industry and therefore representative of the Chilean fish industry. This also includes the presence of some fish of the selected group as IPNv carriers.

During the test, fishes were fed with a diet of 15 micro EWOS 15CP® diet at a daily rate of 0.75% of body weight (bw/day) impregnated with a Quillaja saponaria extract in a dose of 3.75 mg product/Kg of body weight.

Experimental Infection with IPN Virus

Experimental infection with IPNv was performed by intraperitoneal injection in the ventral line at a rate of 0.1 mL of inoculum per fish, with a titer of 1×10⁸ viral copies genome/mL. Additionally, control groups were inoculated with culture medium without virus, in order that all fish were subject to the same management. To perform the inoculation, fish were anaesthetized with benzocaine in a separate reservoir tank at a dose of 40 to 60 ppm. Subsequently, fish recovered in fresh water tank without benzocaine and then were moved into their original experimental tanks, where they stayed during the trial.

Treatment with Quillaja Extracts

The administration was performed using Quillaja extracts given orally for 7 days before infection and then continued feeding with the extracts throughout the post-infection period. Each day, food was prepared using a dose of QD100 product as 3.75 mg of product/Kg body weight (bw) (500 mg product/Kg food), or a dose of UD100 of 3.75 mg product/Kg bw (500 mg product/Kg food), incorporated in 2% fish oil to the pellet. Table 5 shows the test schedule indicated.

TABLE 5 Test schedule of the in vivo efficacy study using Quillaja extracts for the prevention and/or treatment against IPNv infections. Day −10 0 1 7 45 Acclimation Start End Oral treatment with extracts Start End IPN viral inoculation Start

Post-infection follow up Start End Post-treatment follow up Start End Total time of study Start End

indicates data missing or illegible when filed

Subsequently, the efficacy of treatment with Quillaja extracts was evaluated and fish mortality was recorded daily. Table 6 shows the cumulative mortality data.

TABLE 6 Survival percentage of IPNv challenged fish fed with or without Quillaja extracts. Total Final Mortality Mean Mean Tanks fish fish (%) Survival (%) mortality survival (1) Normal diet 20 19 5 95 (2) Normal diet 20 20 0 100 2.5 97.5 (7) Normal diet + IPNV 20 10 50 50 (8) Normal diet + IPNV 20 11 45 55 47.5 52.5 (3) UD100 diet 20 19 5 95 (4) UD100 diet 20 19 5 95 5 95 (9) UD100 + IPNV diet 20 15 25 75 (10) UD100 + IPNV diet 20 18 10 90 17.5 82.5 (5) QD100 diet 20 20 0 100 (6) QD100 diet 20 18 10 90 5 95 (11) QD100 + IPNV diet 20 17 15 85 (12) QD100 + IPNV diet 20 16 20 80 17.5 82.5

Tanks 7, 8, 9, 10, 11 and 12 correspond to studies with fish inoculated with IPNv. The outbreak of mortality occurred between days 18 and 24 post-inoculation. The most mortality was observed in tanks that did not received oral administration of Quillaja extracts. Low mortality was observed in tanks who received oral administration of Quillaja extracts. Tanks with fish treated with Quillaja extracts practically did not develop the disease, compared to that observed in untreated tanks.

Later, all dead fish were necropsied. A non-specific clinical symptom in diseased fish was observed, such as lethargy, dark coloration and death. Microscopically, dead fish showed generally nonspecific signs such as pale necrotic liver and spleen.

The diagnosis was made in IPNv infected dead fish, with positive results for the detection of the virus by amplification by RT-PCR, which confirmed that the fish developed the infection and disease, although in some cases this was moderate.

Importantly, considering the high fish health risk involved in in vivo experimentation assays with ISA virus, and biosecurity protocols such as de duty to eliminate all fish in case of a virus outbreak, and considering the equivalent results obtained in in vitro and in vivo assays for IPNv, taking in consideration the in vitro results obtained for Quillaja extracts in inhibition of viral replication of ISAv from Table 3, which is greater than 99% inmost cases, it is possible to infer that oral administration of these extracts, either as part of a food or a medicinal product, will also confer protection against ISAv to fish.

Thus, the results showed that the use of extracts of Quillaja saponaria for the prevention and/or control against viral diseases in fish is highly effective, which reflect a protective effect against ISA virus and IPN virus with over 90% of effectiveness. Also, it was demonstrated that in vivo efficacy was high for the prevention and/or control of viral infections at a concentration of 0.9375 mg/Kg of total saponins per live weight of fish using Quillaja natural extracts on a controlled infection of Atlantic salmon, achieving a decrease in mortality associated with IPNv of at least 72%. 

1. Use of a Quillaja saponaria extract as active ingredient for the manufacture of a medicinal composition, for prevention and control of viral disease in fish.
 2. The use according to claim 1, wherein the medicinal preparation is administered to the fish orally in a dose range between 0.9 to 12 mg saponins/Kg of live weight of fish.
 3. The use according to claim 2, wherein the administration is carried out orally in combination with fish food.
 4. The use according to claim 1, wherein the viral disease is caused by a virus belonging to the families of the group comprising Birnaviridae, and Orthomyxoviridae.
 5. The use according to claim 4, wherein the virus belonging to the Birnaviridae family belongs to the genus Aquabirnaviridae.
 6. The use according to claim 5, wherein the virus is the infectious pancreatic necrosis virus.
 7. The use according to claim 4, wherein the virus belonging to the Orthomyxoviridae family belongs to the genus Isavirus.
 8. The use according to claim 7, wherein the virus is the virus of the infectious salmon anemia.
 9. The use according to claim 1, wherein the fish are salmonids.
 10. Use according to claim 1, wherein the salmonids are selected from the group consisting of Salmo salar, Salmo trutta, Salmo gairdnerii, Oncorhynchus mykiss or Oncorhynchus kisutch.
 11. A medicinal composition for the prevention and control of viral disease in fish, wherein it comprises a Quillaja saponaria extract as active ingredient and an appropriate excipient.
 12. The composition according to claim 11, wherein the Quillaja saponaria extract is in a ratio of 2 to 90% w/w of the total weight of the composition.
 13. A food composition for the prevention and control of a viral disease in fish, wherein it comprises an extract of Quillaja saponaria as active ingredient and an appropriate excipient.
 14. The composition according to claim 13, wherein the Quillaja saponaria extract is in a proportion of less than 6% w/w of the total composition.
 15. A method for the prevention and control of a viral disease in fish, wherein said method comprises the steps of: providing a medicinal composition comprising a Quillaja saponaria extract as active ingredient; and administering said medicinal composition to the fish in a dose range between 0.9 to 12 mg of saponins/Kg of live weight of fish.
 16. The method according to claim 15, wherein the medicinal preparation is administered to the fish orally in a dose range between 0.9 to 12 mg saponins/Kg of live weight of fish.
 17. The method according to claim 16, wherein the administration is carried out orally in combination with fish food.
 18. The method according to claim 15, wherein the viral disease is caused by a virus belonging to the families of the group comprising Birnaviridae, and Orthomyxoviridae.
 19. The method according to claim 18, wherein the virus belonging to the Birnaviridae family belongs to the genus Aquabirnaviridae.
 20. The method according to claim 19, wherein the virus is the infectious pancreatic necrosis virus.
 21. The method according to claim 18, wherein the virus belonging to the Orthomyxoviridae family belongs to the genus Isavirus.
 22. The method according to claim 21, wherein the virus is the virus of the infectious salmon anemia.
 23. The method according to claim 15, wherein the fish are salmonids.
 24. The method according to claim 23, wherein the salmonids are selected from the group consisting of Salmo salar, Salmo trutta, Salmo gairdnerii, Oncorhynchus mykiss or Oncorhynchus kisutch. 